LOCUS       sequence-380045-       15891 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nimble cloning-compatible vector for genome editing in plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15891)
  AUTHORS   Yan P, Tuo D, Shen W, Deng H, Zhou P, Gao X
  TITLE     A Nimble Cloning-compatible vector system for high-throughput gene 
            functional analysis in plants.
  JOURNAL   Plant Commun. 2023 Mar 13;4(2):100471. doi: 
            10.1016/j.xplc.2022.100471. Epub 2022 Nov 9.
  PUBMED    36352791
REFERENCE   2  (bases 1 to 15891)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Commun."; date: "2023-03-13"; volume: "4(2):100471. doi"; pages: " 
            10.1016/j.xplc.2022.100471. Epub 2022 Nov 9"
COMMENT     Sequence Label: pNC-KSE401
FEATURES             Location/Qualifiers
     source          1..15891
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    21..45
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     233..255
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     247..264
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     248..264
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        879..981
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1012..1317
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1196..1215
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1374..1449
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2063..2407
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             2560..2625
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2632..2652
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2677..6777
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2677..6777
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             6690..6698
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             6778..6825
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(7482..7498)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7482..7498)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7495..7517)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7506..7522
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7530..7536,7537..7554,7555..7560))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7575..7596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7787..8463
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     CDS             8526..9323
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
                     SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
                     QGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
                     ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(8583..8602)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     9193..9212
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    9380..9554
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    9632..9656
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             10081..10875
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      10962..11550
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11451..11470
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11704..11721
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    11736..11876
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(11802..11821)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     11962..11984
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      12220..12414
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(12480..13553)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     CDS             complement(13982..14611)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
ORIGIN
        1 taaacgctct tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta
       61 aactgaaggc gggaaacgac aatctgatcc aagctcaagc tgctctagca ttcgccattc
      121 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      181 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      241 cgacgttgta aaacgacggc cagtgccaag cttcgacttg ccttccgcac aatacatcat
      301 ttcttcttag ctttttttct tcttcttcgt tcatacagtt tttttttgtt tatcagctta
      361 cattttcttg aaccgtagct ttcgttttct tctttttaac tttccattcg gagtttttgt
      421 atcttgtttc atagtttgtc ccaggattag aatgattagg catcgaacct tcaagaattt
      481 gattgaataa aacatcttca ttcttaagat atgaagataa tcttcaaaag gcccctggga
      541 atctgaaaga agagaagcag gcccatttat atgggaaaga acaatagtat ttcttatata
      601 ggcccattta agttgaaaac aatcttcaaa agtcccacat cgcttagata agaaaacgaa
      661 gctgagttta tatacagcta gagtcgaagt agtgattgag tggtctctgt ccagtcctgg
      721 cctcgtcggc cattctcgac taagttggca gcatcacccg acgcactttg cgccgaataa
      781 atacctgtga cggaagatca cttcgcagaa taaataaatc ctggtgtccc tgttgatacc
      841 gggaagccct gggccaactt ttggcgaaaa tgagacgttg atcggcacgt aagaggttcc
      901 aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt atcgagattt
      961 tcaggagcta aggaagctaa acttttgctg acgagaacag ggactggtga aatgcagttt
     1021 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
     1081 attattgaca cgcctgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
     1141 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
     1201 atgaccaccg atatggccag tgtgccggta tccgttatcg gggaagaagt ggctgatctc
     1261 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg
     1321 tcaggctccc ttatacacag ggccagtctg gccacttgtg gtctgctgag accgttttag
     1381 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     1441 agtcggtgct tttttttgca aaattttcca gatcgatttc ttcttcctct gttcttcggc
     1501 gttcaatttc tggggttttc tcttcgtttt ctgtaactga aacctaaaat ttgacctaaa
     1561 aaaaatctca aataatatga ttcagtggtt ttgtactttt cagttagttg agttttgcag
     1621 ttccgatgag ataaaccaat aagcttgcat gcctgcaggt caacatggtg gagcacgaca
     1681 cacttgtcta ctccaaaaat atcaaagata cagtctcaga agaccaaagg gcaattgaga
     1741 cttttcaaca aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc
     1801 actttattgt gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata
     1861 aaggaaaggc catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac
     1921 ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt
     1981 gatgtgataa catggtggag cacgacacac ttgtctactc caaaaatatc aaagatacag
     2041 tctcagaaga ccaaagggca attgagactt ttcaacaaag ggtaatatcc ggaaacctcc
     2101 tcggattcca ttgcccagct atctgtcact ttattgtgaa gatagtggaa aaggaaggtg
     2161 gctcctacaa atgccatcat tgcgataaag gaaaggccat cgttgaagat gcctctgccg
     2221 acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc
     2281 caaccacgtc ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg
     2341 cacaatccca ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg
     2401 agaggacctc gacctcaaca caacatatac aaaacaaacg aatctcaagc aatcaagcat
     2461 tctacttcta ttgcagcaat ttaaatcatt tcttttaaag caaaagcaat tttctgaaaa
     2521 ttttcaccat ttacgaacga tactcgagta atctagatgg attacaagga ccacgacggg
     2581 gattacaagg accacgacat tgattacaag gatgatgatg acaagatggc tccgaagaag
     2641 aagaggaagg ttggcatcca cggggtgcca gctgctgaca agaagtactc gatcggcctc
     2701 gatattggga ctaactctgt tggctgggcc gtgatcaccg acgagtacaa ggtgccctca
     2761 aagaagttca aggtcctggg caacaccgat cggcattcca tcaagaagaa tctcattggc
     2821 gctctcctgt tcgacagcgg cgagacggct gaggctacgc ggctcaagcg caccgcccgc
     2881 aggcggtaca cgcgcaggaa gaatcgcatc tgctacctgc aggagatttt ctccaacgag
     2941 atggcgaagg ttgacgattc tttcttccac aggctggagg agtcattcct cgtggaggag
     3001 gataagaagc acgagcggca tccaatcttc ggcaacattg tcgacgaggt tgcctaccac
     3061 gagaagtacc ctacgatcta ccatctgcgg aagaagctcg tggactccac agataaggcg
     3121 gacctccgcc tgatctacct cgctctggcc cacatgatta agttcagggg ccatttcctg
     3181 atcgaggggg atctcaaccc ggacaatagc gatgttgaca agctgttcat ccagctcgtg
     3241 cagacgtaca accagctctt cgaggagaac cccattaatg cgtcaggcgt cgacgcgaag
     3301 gctatcctgt ccgctaggct ctcgaagtct cggcgcctcg agaacctgat cgcccagctg
     3361 ccgggcgaga agaagaacgg cctgttcggg aatctcattg cgctcagcct ggggctcacg
     3421 cccaacttca agtcgaattt cgatctcgct gaggacgcca agctgcagct ctccaaggac
     3481 acatacgacg atgacctgga taacctcctg gcccagatcg gcgatcagta cgcggacctg
     3541 ttcctcgctg ccaagaatct gtcggacgcc atcctcctgt ctgatattct cagggtgaac
     3601 accgagatta cgaaggctcc gctctcagcc tccatgatca agcgctacga cgagcaccat
     3661 caggatctga ccctcctgaa ggcgctggtc aggcagcagc tccccgagaa gtacaaggag
     3721 atcttcttcg atcagtcgaa gaacggctac gctgggtaca ttgacggcgg ggcctctcag
     3781 gaggagttct acaagttcat caagccgatt ctggagaaga tggacggcac ggaggagctg
     3841 ctggtgaagc tcaatcgcga ggacctcctg aggaagcagc ggacattcga taacggcagc
     3901 atcccacacc agattcatct cggggagctg cacgctatcc tgaggaggca ggaggacttc
     3961 taccctttcc tcaaggataa ccgcgagaag atcgagaaga ttctgacttt caggatcccg
     4021 tactacgtcg gcccactcgc taggggcaac tcccgcttcg cttggatgac ccgcaagtca
     4081 gaggagacga tcacgccgtg gaacttcgag gaggtggtcg acaagggcgc tagcgctcag
     4141 tcgttcatcg agaggatgac gaatttcgac aagaacctgc caaatgagaa ggtgctccct
     4201 aagcactcgc tcctgtacga gtacttcaca gtctacaacg agctgactaa ggtgaagtat
     4261 gtgaccgagg gcatgaggaa gccggctttc ctgtctgggg agcagaagaa ggccatcgtg
     4321 gacctcctgt tcaagaccaa ccggaaggtc acggttaagc agctcaagga ggactacttc
     4381 aagaagattg agtgcttcga ttcggtcgag atctctggcg ttgaggaccg cttcaacgcc
     4441 tccctgggga cctaccacga tctcctgaag atcattaagg ataaggactt cctggacaac
     4501 gaggagaatg aggatatcct cgaggacatt gtgctgacac tcactctgtt cgaggaccgg
     4561 gagatgatcg aggagcgcct gaagacttac gcccatctct tcgatgacaa ggtcatgaag
     4621 cagctcaaga ggaggaggta caccggctgg gggaggctga gcaggaagct catcaacggc
     4681 attcgggaca agcagtccgg gaagacgatc ctcgacttcc tgaagagcga tggcttcgcg
     4741 aaccgcaatt tcatgcagct gattcacgat gacagcctca cattcaagga ggatatccag
     4801 aaggctcagg tgagcggcca gggggactcg ctgcacgagc atatcgcgaa cctcgctggc
     4861 tcgccagcta tcaagaaggg gattctgcag accgtgaagg ttgtggacga gctggtgaag
     4921 gtcatgggca ggcacaagcc tgagaacatc gtcattgaga tggcccggga gaatcagacc
     4981 acgcagaagg gccagaagaa ctcacgcgag aggatgaaga ggatcgagga gggcattaag
     5041 gagctggggt cccagatcct caaggagcac ccggtggaga acacgcagct gcagaatgag
     5101 aagctctacc tgtactacct ccagaatggc cgcgatatgt atgtggacca ggagctggat
     5161 attaacaggc tcagcgatta cgacgtcgat catatcgttc cacagtcatt cctgaaggat
     5221 gactccattg acaacaaggt cctcaccagg tcggacaaga accggggcaa gtctgataat
     5281 gttccttcag aggaggtcgt taagaagatg aagaactact ggcgccagct cctgaatgcc
     5341 aagctgatca cgcagcggaa gttcgataac ctcacaaagg ctgagagggg cgggctctct
     5401 gagctggaca aggcgggctt catcaagagg cagctggtcg agacacggca gatcactaag
     5461 cacgttgcgc agattctcga ctcacggatg aacactaagt acgatgagaa tgacaagctg
     5521 atccgcgagg tgaaggtcat caccctgaag tcaaagctcg tctccgactt caggaaggat
     5581 ttccagttct acaaggttcg ggagatcaac aattaccacc atgcccatga cgcgtacctg
     5641 aacgcggtgg tcggcacagc tctgatcaag aagtacccaa agctcgagag cgagttcgtg
     5701 tacggggact acaaggttta cgatgtgagg aagatgatcg ccaagtcgga gcaggagatt
     5761 ggcaaggcta ccgccaagta cttcttctac tctaacatta tgaatttctt caagacagag
     5821 atcactctgg ccaatggcga gatccggaag cgccccctca tcgagacgaa cggcgagacg
     5881 ggggagatcg tgtgggacaa gggcagggat ttcgcgaccg tcaggaaggt tctctccatg
     5941 ccacaagtga atatcgtcaa gaagacagag gtccagactg gcgggttctc taaggagtca
     6001 attctgccta agcggaacag cgacaagctc atcgcccgca agaaggactg ggatccgaag
     6061 aagtacggcg ggttcgacag ccccactgtg gcctactcgg tcctggttgt ggcgaaggtt
     6121 gagaagggca agtccaagaa gctcaagagc gtgaaggagc tgctggggat cacgattatg
     6181 gagcgctcca gcttcgagaa gaacccgatc gatttcctgg aggcgaaggg ctacaaggag
     6241 gtgaagaagg acctgatcat taagctcccc aagtactcac tcttcgagct ggagaacggc
     6301 aggaagcgga tgctggcttc cgctggcgag ctgcagaagg ggaacgagct ggctctgccg
     6361 tccaagtatg tgaacttcct ctacctggcc tcccactacg agaagctcaa gggcagcccc
     6421 gaggacaacg agcagaagca gctgttcgtc gagcagcaca agcattacct cgacgagatc
     6481 attgagcaga tttccgagtt ctccaagcgc gtgatcctgg ccgacgcgaa tctggataag
     6541 gtcctctccg cgtacaacaa gcaccgcgac aagccaatca gggagcaggc tgagaatatc
     6601 attcatctct tcaccctgac gaacctcggc gcccctgctg ctttcaagta cttcgacaca
     6661 actatcgatc gcaagaggta cacaagcact aaggaggtcc tggacgcgac cctcatccac
     6721 cagtcgatta ccggcctcta cgagacgcgc atcgacctgt ctcagctcgg gggcgacaag
     6781 cggccagcgg cgacgaagaa ggcggggcag gcgaagaaga agaagtgagc tcagagcttt
     6841 cgttcgtatc atcggtttcg acaacgttcg tcaagttcaa tgcatcagtt tcattgcgca
     6901 cacaccagaa tcctactgag tttgagtatt atggcattgg gaaaactgtt tttcttgtac
     6961 catttgttgt gcttgtaatt tactgtgttt tttattcggt tttcgctatc gaactgtgaa
     7021 atggaaatgg atggagaaga gttaatgaat gatatggtcc ttttgttcat tctcaaatta
     7081 atattatttg ttttttctct tatttgttgt gtgttgaatt tgaaattata agagatatgc
     7141 aaacattttg ttttgagtaa aaatgtgtca aatcgtggcc tctaatgacc gaagttaata
     7201 tgaggagtaa aacacttgta gttgtaccat tatgcttatt cactaggcaa caaatatatt
     7261 ttcagaccta gaaaagctgc aaatgttact gaatacaagt atgtcctctt gtgttttaga
     7321 catttatgaa ctttccttta tgtaattttc cagaatcctt gtcagattct aatcattgct
     7381 ttataattat agttatactc atggatttgt agttgagtat gaaaatattt tttaatgcat
     7441 tttatgactt gccaattgat tgacaacgaa ttcgtaatca tgtcatagct gtttcctgtg
     7501 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     7561 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     7621 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     7681 ggcggtttgc gtattggcta gagcagcttg ccaacatggt ggagcacgac actctcgtct
     7741 actccaagaa tatcaaagat acagtctcag aagaccaaag ggctattgag acttttcaac
     7801 aaagggtaat atcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatca
     7861 aaaggacagt agaaaaggaa ggtggcacct acaaatgcca tcattgcgat aaaggaaagg
     7921 ctatcgttca agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga
     7981 gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgaac
     8041 atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt ctcagaagac
     8101 caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct cggattccat
     8161 tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg cacctacaaa
     8221 tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga cagtggtccc
     8281 aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct
     8341 tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc acaatcccac
     8401 tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga gaggacacgc
     8461 tgaaatcacc agtctctctc tacaaatcta tctctctcga gctttcgcag atctgtcgat
     8521 cgaccatggg gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     8581 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgtttc
     8641 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     8701 atgaactcca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     8761 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     8821 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     8881 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     8941 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     9001 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     9061 tgcccgacgg cgaggatctc gtcgtgacac atggcgatgc ctgcttgccg aatatcatgg
     9121 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     9181 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     9241 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     9301 gccttcttga cgagttcttc tgagcgggac tctggggttc ggatcgatcc tctagctaga
     9361 gtcgatcgac aagctcgagt ttctccataa taatgtgtga gtagttccca gataagggaa
     9421 ttagggttcc tatagggttt cgctcatgtg ttgagcatat aagaaaccct tagtatgtat
     9481 ttgtatttgt aaaatacttc tatcaataaa atttctaatt cctaaaacca aaatccagta
     9541 ctaaaatcca gatcccccga attaattcgg cgttaattca gtacattaaa aacgtccgca
     9601 atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag
     9661 ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat acaggcagcc
     9721 catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagact ttgctcatgt
     9781 taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacac ggatgatctc
     9841 gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg atcaccgcgg
     9901 tttcaaaatc ggctccgtcg atactatgtt atacgccaac tttgaaaaca actttgaaaa
     9961 agctgttttc tggtatttaa ggttttagaa tgcaaggaac agtgaattgg agttcgtctt
    10021 gttataatta gcttcttggg gtatctttaa atactgtaga aaagaggaag gaaataataa
    10081 atggctaaaa tgagaatatc accggaattg aaaaaactga tcgaaaaata ccgctgcgta
    10141 aaagatacgg aaggaatgtc tcctgctaag gtatataagc tggtgggaga aaatgaaaac
    10201 ctatatttaa aaatgacgga cagccggtat aaagggacca cctatgatgt ggaacgggaa
    10261 aaggacatga tgctatggct ggaaggaaag ctgcctgttc caaaggtcct gcactttgaa
    10321 cggcatgatg gctggagcaa tctgctcatg agtgaggccg atggcgtcct ttgctcggaa
    10381 gagtatgaag atgaacaaag ccctgaaaag attatcgagc tgtatgcgga gtgcatcagg
    10441 ctctttcact ccatcgacat atcggattgt ccctatacga atagcttaga cagccgctta
    10501 gccgaattgg attacttact gaataacgat ctggccgatg tggattgcga aaactgggaa
    10561 gaagacactc catttaaaga tccgcgcgag ctgtatgatt ttttaaagac ggaaaagccc
    10621 gaagaggaac ttgtcttttc ccacggcgac ctgggagaca gcaacatctt tgtgaaagat
    10681 ggcaaagtaa gtggctttat tgatcttggg agaagcggca gggcggacaa gtggtatgac
    10741 attgccttct gcgtccggtc gatcagggag gatatcgggg aagaacagta tgtcgagcta
    10801 ttttttgact tactggggat caagcctgat tgggagaaaa taaaatatta tattttactg
    10861 gatgaattgt tttagtacct agaatgcatg accaaaatcc cttaacgtga gttttcgttc
    10921 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
    10981 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
    11041 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
    11101 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
    11161 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
    11221 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
    11281 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
    11341 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
    11401 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
    11461 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
    11521 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
    11581 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
    11641 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
    11701 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
    11761 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
    11821 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
    11881 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
    11941 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
    12001 cgaaacgcgc gaggcagggt gccttgatgt gggcgccggc ggtcgagtgg cgacggcgcg
    12061 gcttgtccgc gccctggtag attgcctggc cgtaggccag ccatttttga gcggccagcg
    12121 gccgcgatag gccgacgcga agcggcgggg cgtagggagc gcagcgaccg aagggtaggc
    12181 gctttttgca gctcttcggc tgtgcgctgg ccagacagtt atgcacaggc caggcgggtt
    12241 ttaagagttt taataagttt taaagagttt taggcggaaa aatcgccttt tttctctttt
    12301 atatcagtca cttacatgtg tgaccggttc ccaatgtacg gctttgggtt cccaatgtac
    12361 gggttccggt tcccaatgta cggctttggg ttcccaatgt acgtgctatc cacaggaaac
    12421 agaccttttc gacctttttc ccctgctagg gcaatttgcc ctagcatctg ctccgtacat
    12481 taggaaccgg cggatgcttc gccctcgatc aggttgcggt agcgcatgac taggatcggg
    12541 ccagcctgcc ccgcctcctc cttcaaatcg tactccggca ggtcatttga cccgatcagc
    12601 ttgcgcacgg tgaaacagaa cttcttgaac tctccggcgc tgccactgcg ttcgtagatc
    12661 gtcttgaaca accatctggc ttctgccttg cctgcggcgc ggcgtgccag gcggtagaga
    12721 aaacggccga tgccgggatc gatcaaaaag taatcggggt gaaccgtcag cacgtccggg
    12781 ttcttgcctt ctgtgatctc gcggtacatc caatcagcta gctcgatctc gatgtactcc
    12841 ggccgcccgg tttcgctctt tacgatcttg tagcggctaa tcaaggcttc accctcggat
    12901 accgtcacca ggcggccgtt cttggccttc ttcgtacgct gcatggcaac gtgcgtggtg
    12961 tttaaccgaa tgcaggtttc taccaggtcg tctttctgct ttccgccatc ggctcgccgg
    13021 cagaacttga gtacgtccgc aacgtgtgga cggaacacgc ggccgggctt gtctcccttc
    13081 ccttcccggt atcggttcat ggattcggtt agatgggaaa ccgccatcag taccaggtcg
    13141 taatcccaca cactggccat gcccgccggc cctgcggaaa cctctacgtg cccgtctgga
    13201 agctcgtagc ggatcacctc gccagctcgt cggtcacgct tcgacagacg gaaaacggcc
    13261 acgtccatga tgctgcgact atcgcgggtg cccacgtcat agagcatcgg aacgaaaaaa
    13321 tctggttgct cgtcgccctt gggcggcttc ctaatcgacg gcgcaccggc tgccggcggt
    13381 tgccgggatt ctttgcggat tcgatcagcg gccgcttgcc acgattcacc ggggcgtgct
    13441 tctgcctcga tgcgttgccg ctgggcggcc tgcgccgcct tcaacttctc caccaggtca
    13501 tcacccagcg ccgcgccgat ttgtaccggg ccggatggtt tgcgaccgct cacgccgatt
    13561 cctcgggctt gggggttcca gtgccattgc agggccggca ggcaacccag ccgcttacgc
    13621 ctggccaacc gcccgttcct ccacacatgg ggcattccac ggcgtcggtg cctggttgtt
    13681 cttgattttc catgccgcct cctttagccg ctaaaattca tctactcatt tattcatttg
    13741 ctcatttact ctggtagctg cgcgatgtat tcagatagca gctcggtaat ggtcttgcct
    13801 tggcgtaccg cgtacatctt cagcttggtg tgatcctccg ccggcaactg aaagttgacc
    13861 cgcttcatgg ctggcgtgtc tgccaggctg gccaacgttg cagccttgct gctgcgtgcg
    13921 ctcggacggc cggcacttag cgtgtttgtg cttttgctca ttttctcttt acctcattaa
    13981 ctcaaatgag ttttgattta atttcagcgg ccagcgcctg gacctcgcgg gcagcgtcgc
    14041 cctcgggttc tgattcaaga acggttgtgc cggcggcggc agtgcctggg tagctcacgc
    14101 gctgcgtgat acgggactca agaatgggca gctcgtaccc ggccagcgcc tcggcaacct
    14161 caccgccgat gcgcgtgcct ttgatcgccc gcgacacgac aaaggccgct tgtagccttc
    14221 catccgtgac ctcaatgcgc tgcttaacca gctccaccag gtcggcggtg gcccatatgt
    14281 cgtaagggct tggctgcacc ggaatcagca cgaagtcggc tgccttgatc gcggacacag
    14341 ccaagtccgc cgcctggggc gctccgtcga tcactacgaa gtcgcgccgg ccgatggcct
    14401 tcacgtcgcg gtcaatcgtc gggcggtcga tgccgacaac ggttagcggt tgatcttccc
    14461 gcacggccgc ccaatcgcgg gcactgccct ggggatcgga atcgactaac agaacatcgg
    14521 ccccggcgag ttgcagggcg cgggctagat gggttgcgat ggtcgtcttg cctgacccgc
    14581 ctttctggtt aagtacagcg ataaccttca tgcgttcccc ttgcgtattt gtttatttac
    14641 tcatcgcatc atatacgcag cgaccgcatg acgcaagctg ttttactcaa atacacatca
    14701 cctttttaga cggcggcgct cggtttcttc agcggccaag ctcgccggcc aggccgccag
    14761 cttggcatca gacaaaccgg ccaggatttc atgcagccgc acggttgaga cgtgcgcggg
    14821 cggctcgaac acgtacccgg ccgcgatcat ctccgcctcg atctcttcgg taatgaaaaa
    14881 cggttcgtcc tggccgtcct ggtgcggttt catgcttgtt cctcttggcg ttcattctcg
    14941 gcggccgcca gggcgtcggc ctcggtcaat gcgtcctcac ggaaggcacc gcgccgcctg
    15001 gcctcggtgg gcgtcacttc ctcgctgcgc tcaagtgcgc ggtacagggt cgagcgatgc
    15061 acgccaagca gtgcagccgc ctctttcacg gtgcggcctt cctggtcgat cagctcgcgg
    15121 gcgtgcgcga tctgtgccgg ggtgagggta gggcgggggc caaacttcac gcctcgcgcc
    15181 ttggcggcct cgcgcccgct ccgggtgcgg tcgatgatta gggaacgctc gaactcggca
    15241 atgccggcga acacggtcaa caccatgcgg ccggccggcg tggtggtgtc ggcccacggc
    15301 tctgccaggc tacgcaggcc cgcgccggcc tcctggatgc gctcggcaat gtccagtagg
    15361 tcgcgggtgc tgcgggccag gcggtctagc ctggtcactg tcacaacgtc gccagggcgt
    15421 aggtggtcaa gcatcctggc cagctccggg cggtcgcgcc tggtgccggt gatcttctcg
    15481 gaaaacagct tggtgcagcc ggccgcgtgc agttcggccc gttggttggt caagtcctgg
    15541 tcgtcggtgc tgacgcgggc atagcccagc aggccagcgg cggcgctctt gttcatggcg
    15601 taatgtctcc ggttctagtc gcaagtattc tactttatgc gactaaaaca cgcgacaaga
    15661 aaacgccagg aaaagggcag ggcggcagcc tgtcgcgtaa cttaggactt gtgcgacatg
    15721 tcgttttcag aagacggctg cactgaacgt cagaagccga ctgcactata gcagcggagg
    15781 ggttggatca aagtactttg atcccgaggg gaaccctgtg gttggcatgc acatacaaat
    15841 ggacgaacgg ataaaccttt tcacgccctt ttaaatatcc gattattcta a
//
